Phosphoinositide-based switches in endocytic membrane traffic and signaling.
Phosphoinositides (PIPs) regulate nearly all aspects of cell physiology ranging from cell signaling to membrane compartmentalization and organelle dynamics. A hallmark of PIP function is their rapid interconversion by PIP kinases and phosphatases, which underlies PIP-based functional switches in endolysosomal membrane traffic and in cell signaling. How PIP switches operate in space and time in most cases is unknown. In the past funding period we have focused on the analysis of PIP-based functional switches that operate during endocytosis at the plasma membrane and within the endolysosomal system to locally regulate nutrient signaling.
The Schultz lab completed the synthesis of a novel tool set of lipid derivatives that we will use in the forthcoming funding period to identify novel effectors of PI 3-phosphates in endocytosis and at endolysosomal compartments. Major efforts in the Haucke lab have been devoted to the manipulation of different PI 3- phosphates at the plasma membrane. We could identify an autoregulatory lipid switch that controls the phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]-synthesizing activity of PI3KC2α during clathrin-mediated endocytosis (CME) at the cell surface (Wang et al., Mol Cell 2018). Furthermore, we have combined genetic knockdown/ rescue and CRISPR knockout (KO) approaches with the application of membrane-permeant short and long chain PIP derivatives, and acute rapalog-mediated depletion of PIPs to further dissect the role of PI 3-phosphates in CME. These experiments have delineated a compartment specific cascade of PIP conversion reactions from the plasma membrane en route to the endolysosomal system. In a third set of studies we discovered PI3KC2β as a local repressor or nutrient signaling by mTORC1 at late endosomes and lysosomes (Marat et al Science 2017). Using quantitative proteomic, biochemical, and cell biological approaches we have identified protein kinase N (PKN) and the mTORC2 complex as upstream regulators that control the PI3KC2β/ PI(3,4)P2-based lysosomal lipid switch to integrate cellular nutrient status with lysosome function (Wallroth et al., Nat Cell Biol, 2019).
In the forthcoming funding period, we will capitalize on these findings and the novel lipid tools made by the Schultz lab to dissect the role of PI 3-phosphates as central regulatory switches that control lysosome biology. In aim 1 we will (i) identify and characterize novel PI 3-phosphate-binding effector proteins recruited to lysosomal membrane compartments in response to alterations in cellular nutrient status. Furthermore, we will analyze the mechanisms by which distinct PI 3-phosphates and their effectors regulate lysosomal nutrient signaling and protein turnover (aim 2), and how this is coupled to the control of lysosome position and dynamics (aim 3). We anticipate that these studies will provide new insights into the mechanisms underlying PIP-based functional switches at lysosomes and how dysfunction of such switches may contribute to disease.